Fakultät für Medizin Institut für Virologie
Molecular Mechanisms of Cytokine-induced, Non-cytolytic Control of Hepatitis B Virus Persistence
Daniela Stadler
Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation.
Vorsitzender: Prof. Dr. Dirk H. Busch
Prüfende der Dissertation:
1. Prof. Dr. Ulrike Protzer 2. Prof. Dr. Bernhard Küster 3. Prof. Dr. Oliver T. Keppler
Die Dissertation wurde am 17.05.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 06.12.2017 angenommen.
Abbreviations ... 7
Abstract ...11
Zusammenfassung ...13
1 Introduction ...15
1.1 Hepatitis B virus (HBV) ...15
1.1.1 Classification of HBV ...15
1.1.2 Structure of HBV particles ...16
1.1.3 HBV genome organisation ...16
1.1.4 Replication cycle of HBV ...17
1.1.5 Formation and persistence of HBV cccDNA ...20
1.1.6 HBV infection and therapy ...22
1.2 Antiviral T-cell immunity ...23
1.2.1 T-cell responses in acute and chronic hepatitis B ...23
1.2.2 Cytotoxic T-cell response ...23
1.2.3 Non-cytolytic T-cell functions ...24
1.3 Antiviral effects of cytokines ...25
1.3.1 Interferon-alpha (IFN-) ...25
1.3.2 Interferon-gamma (IFN-) ...26
1.3.3 Tumour necrosis factor-alpha (TNF-) ...27
1.3.4 Lymphotoxins (LT) ...28
1.3.5 APOBEC3 deaminases as cytokine-induced effector proteins ...30
1.3.6 ISG20 as interferon-induced effector nuclease ...31
1.4 Aims of the study ...32
2 Results ...33
2.1 Non-cytolytic reduction of HBV cccDNA by T-cell cytokines IFN- and TNF-...33
2.1.1 Activated T cells secrete IFN- and TNF- and induce cccDNA loss ....33
2.1.2 Decline of cccDNA is mediated by T-cell cytokines IFN- and TNF- ...36
2.1.3 Cytokine-induced A3A and A3B are essential for cccDNA deamination
and decay ... 38
2.2 Specificity of treatment-induced cccDNA loss... 40
2.2.1 Treatment-induced decay and deamination targets cccDNA but not episomal DNA... 40
2.2.2 A3A and A3B induce hypermutations on cccDNA ... 43
2.2.3 A3A and HBV core protein bind to cccDNA ... 44
2.3 Identification of ISG20 as the nuclease in interferon-induced decline of cccDNA ... 45
2.3.1 A3A leads to deamination but is not sufficient for cccDNA reduction ... 45
2.3.2 Interferons upregulate expression of the nuclease ISG20 ... 48
2.3.3 ISG20 is expressed in acute hepatitis B and in co-infections ... 51
2.3.4 ISG20 knockdown abolishes interferon-induced loss of cccDNA ... 54
2.3.5 ISG20 overexpression together with A3A is sufficient for cccDNA reduction ... 58
3 Discussion ... 61
3.1 Non-cytolytic reduction of HBV cccDNA by T-cell cytokines IFN- and TNF- ... 61
3.1.1 IFN- and TNF- as the main T-cell cytokines mediating cccDNA loss 61 3.1.2 Impact of T-cell cytokines IFN- and TNF- on controlling HBV infection in patients ... 62
3.1.3 Non-cytolytic cccDNA reduction ... 62
3.1.4 Cytokine signalling diminishes cccDNA independent of rcDNA reimport... 63
3.1.5 Physiological relevance of non-cytolytic T-cell cytokines in comparison to T-cell killing ... 64
3.1.6 Therapeutic implications of non-cytolytic T-cell functions ... 64
3.2 Specificity of treatment-induced cccDNA loss... 65
3.2.1 Role of the HBV core protein for cccDNA targeting ... 65
3.2.2 Absence of deamination of genomic DNA by APOBEC3 proteins ... 66
3.2.3 Fate of deaminated HBV DNA: repair versus degradation ... 67
3.2.4 Epigenetic impact on the accessibility of cccDNA ... 67
3.3.1 Damaged cccDNA as substrate for ISG20 ...68
3.3.2 Reduction of cccDNA through ISG20 is independent of nuclear reimport …...……….69
3.3.3 Potential involvement of additional nucleases in cccDNA decay ...69
3.3.4 Impact of ISG20 on HBV clearance in patients ...70
3.3.5 Decline of cccDNA by concerted expression of A3A and ISG20 ...70
3.4 Conclusions ...71
4 Materials and methods ...73
4.1 Materials ...73
4.1.1 Cell lines ...73
4.1.2 Cell culture media ...73
4.1.3 Oligonucleotides for PCR ...74
4.1.4 Kits ...75
4.1.5 Antibodies...76
4.1.6 Plasmids ...76
4.1.7 Chemicals and reagents ...76
4.1.8 Laboratory equipment and consumables ...78
4.1.9 Software ...80
4.2 Methods ...80
4.2.1 Cell culture and treatments ...80
4.2.2 T-cell transwell co-culture system ...81
4.2.3 Enzyme-linked immunosorbent assay (ELISA) ...82
4.2.4 HBV production and infection ...82
4.2.5 DNA extraction ...83
4.2.6 Measurement of HBV markers and pEpi-H1.3 ...83
4.2.7 Differential DNA denaturation PCR (3D-PCR) and sequence analysis .84 4.2.8 Cytotoxicity assay: LDH release assay ...85
4.2.9 Knockdowns of A3A, A3B and ISG20 by siRNA ...85
4.2.10 RNA extractionandquantitative reverse transcription PCR(qRT-PCR) 86 4.2.11 Plasmid transfection ...86
4.2.12 Chromatin immunoprecipitation (ChIP) ... 86
4.2.13 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses ... 87
4.2.14 Microarray-based gene expression analysis ... 88
4.2.15 Immunohistochemistry ... 88
4.2.16 Adenoviral transduction ... 88
4.2.17 Statistical analysis ... 89
5 Figures ... 91
6 References ... 93
Acknowledgement ... 105
Publications and meetings ... 107
Abbreviations
3D-PCR differential DNA denaturation-PCR
A adenine
A3A APOBEC3A
A3B APOBEC3B
A3G APOBEC3G
AdV adenovirus, adenoviral vector
AID activation-induced cytidine deaminase
ALT alanine aminotransferase
AP site apurinic/apyrimidinic site
APC antigen presenting cell
APEX apurinic/apyrimidinic site endonuclease APOBEC3 apolipoprotein B editing complex 3
APS ammonium persulfate
BS1 LTR agonist (bispecific antibody)
C cytosine
cccDNA covalently closed circular DNA
ChIP chromatin immunoprecipitation; Chromatin-Immunopräzipitation cIAP cellular inhibitor of apoptosis
CMV cytomegalovirus
c-myc myelocytomatosis gene
DAB 3,3’-diaminobenzidine
DHBV duck hepatitis B virus
DIG digoxigenin
DMSO dimethyl sulfoxide
DNase deoxyribonuclease
dpi days post-infection
DR1 direct repeat 1
DR2 direct repeat 2
DZIF Deutsches Zentrum für Infektionsforschung ELISA Enzyme-linked immunosorbent assay FADD Fas-associated death domain
FasL Fas ligand
FBS fetal bovine serum
FCS fetal calf serum
FRET fluorescence resonance energy transfer
G guanine
GAF IFN- activation factor
GAPDH glyceraldehyde-3-phosphate dehydrogenase gene
GAS gamma-activated sequence
GFP green fluorescent protein
HBc HBV core protein
HBeAg hepatitis B e antigen
HBs HBV surface protein
HBsAg hepatitis B surface antigen
HBV hepatitis B virus; Hepatitis-B-Virus
HBx HBV X protein
HCV hepatitis C virus
HDV hepatitis D virus
HEM45 HeLa Estrogen Modulated, band 45 HIV-1 human immunodeficiency virus type 1
IFNAR1 IFN- receptor 1
IFNAR2 IFN- receptor 2
IFNGR IFN- receptor
IFNLR1 interferon-lambda receptor 1
IFN- interferon-alpha
IFN- interferon-beta
IFN- interferon-gamma
IL-10R2 interleukin-10 receptor 2
IPTG isopropyl--D-thiogalactopyranosid IRF9 interferon-regulatory factor 9 ISG interferon-stimulated gene
ISG20 interferon-stimulated gene product of 20 kDa ISGF3 interferon-stimulated gene factor 3
ISRE interferon-stimulated response element
IU infectious units
IB inhibitor of B
JAK1 Janus kinase 1
L protein, LHBs large HBV surface protein
LB Luria-Bertani
LDH lactate dehydrogenase
LT lymphotoxin-alpha
LT lymphotoxin-beta
LTR lymphotoxin receptor; Lymphotoxin--Rezeptor M protein, MHBs middle HBV surface protein
MHC I major histocompatibility complex I
MNase micrococcal nuclease
MOI multiplicity of infection
mRNA messenger RNA
MxA myxoma resistance protein 1
nd not detectable
NIK NF-B-inducing kinase
ns not significant
NTCP sodium taurocholate cotransporting polypeptide
P protein polymerase protein of HBV (reverse transcriptase, RNaseH, primer)
p53 tumour suppressor p53 gene
PBS phosphate buffered saline
PBS-T phosphate buffered saline with Tween 20
PCR polymerase chain reaction
PD-1 programmed death-1
PEG polyethylene glycol
PELO protein pelota homolog gene
pgRNA pregenomic RNA
PHH primary human hepatocytes
PLA proximity ligation assay
PNPT1 human polynucleotide phosphorylase (hPNPase) gene
POLK DNA polymerase
PRNP prion protein gene
PTM post-translational modification PVDF polyvinylidene fluoride
qPCR quantitative PCR
qRT-PCR quantitative reverse transcription PCR RAD9A RAD9 checkpoint clamp component A gene rcDNA relaxed-circular DNA
RIN RNA integrity number
RNA pol II RNA polymerase II
RNase ribonuclease
S protein, SHBs small HBV surface protein
S-CAR chimeric antigen receptor recognizing HBsAg
SDS sodium dodecyl sulphate
SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis
shRNA short hairpin RNA
siRNA small interfering RNA
Src cellular/sarcoma tyrosine kinase gene SSC saline-sodium citrate (buffer)
STAT1 signal transducers and activators of transcription 1 STAT2 signal transducers and activators of transcription 2
T thymine
TBP TATA-box binding protein gene
TBS-T Tris-buffered saline with Tween 20
TCR T-cell receptor
TDP tyrosyl-DNA-phosphodiesterase
TEMED tetramethylethylenediamine
Tet tetracycline
TNFR1 TNF receptor 1
TNFR2 TNF receptor 2
TNF- tumour necrosis factor-alpha; Tumornekrosefaktor-alpha
TR tetracycline repressor
TRADD TNFR1-associated death domain
TYK2 tyrosine kinase 2
U unit
UNG uracil DNA glycosylase
WT wild type
X-gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside XRN1 5’ to 3’ exoribonuclease 1 gene
XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5- Carboxanilide
Abstract
Despite an effective vaccine, an estimated 257 million humans worldwide are suffering from chronic hepatitis B resulting in more than 800,000 deaths yearly. Current treatments for chronic hepatitis B can control the replication of the hepatitis B virus (HBV) but cannot eradicate it completely due to the persistence of the covalently closed circular DNA (cccDNA) form of HBV. The cccDNA is therefore a major therapeutic target. Thus, the aim of this thesis was to elucidate the mechanisms of non-cytolytic cccDNA purging through cytokines in more detail.
In the first part, transwell co-culture and cytokine neutralization experiments demonstrated that activated T-cells secrete the cytokines interferon-gamma (IFN-) and tumour necrosis factor-alpha (TNF-), which mediated the reduction of cccDNA in HBV- infected hepatoma cells without cytolysis. These cytokines induced the expression of APOBEC3A (A3A) and APOBEC3B (A3B), that deaminated nuclear HBV DNA. A3A and A3B were essential for cytokine-triggered cccDNA loss as shown by knockdown experiments.
In the second part, the specificity of cccDNA reduction was studied. Only cccDNA but not an episomal HBV DNA construct (pEpi-H1.3) was affected by cytokine-induced deamination and decay through T-cell cytokines, interferon-alpha (IFN-) or lymphotoxin
receptor (LTR) activation. Since all these treatments trigger the expression of A3A or A3B, these deaminases were overexpressed in HBV-replicating hepatoma cells leading to hypermutations on cccDNA. Furthermore, A3A and HBV core protein were shown to associate with cccDNA by chromatin immunoprecipitation (ChIP) experiments, supporting the hypothesis of HBV-core-assisted targeting of nuclear APOBEC3 proteins to cccDNA.
Since A3A expression in human hepatoma cells led to deamination but was not sufficient for cccDNA reduction itself, the third part of this thesis aimed at identifying the nuclease involved in cccDNA purging. Gene expression profiling under IFN- treatment revealed three upregulated candidate nucleases with nuclear localization. ISG20 (interferon- stimulated gene product of 20 kDa), the strongest interferon-induced nuclease, was stained immunohistochemically in liver tissue samples from patients with acute but not chronic hepatitis B. Knockdown of ISG20 rescued the interferon-induced cccDNA loss.
Overexpression of ISG20 together with A3A was sufficient to reduce cccDNA without further treatment.
Overall, the results of this thesis lead to the conclusion that T-cell cytokines IFN- and TNF- as well as IFN- and LTR activation lead to the non-cytolytic purging of cccDNA, the HBV persistence form, from the nucleus of infected hepatocytes involving APOBEC3 family deaminases and the interferon-induced nuclease ISG20. Concerted expression of the deaminase A3A and the nuclease ISG20 suffices to reduce cccDNA, providing a relevant basis for advancing therapies of chronic hepatitis B.
Zusammenfassung
Trotz einer wirksamen Impfung leiden weltweit geschätzte 257 Millionen Menschen an chronischer Hepatitis B, dies führt jährlich zu mehr als 800.000 Todesfällen. Derzeitige Therapien können die Replikation des Hepatitis-B-Virus (HBV) zwar hemmen, führen aber nicht zu einer vollständigen Eliminierung des Virus aufgrund der Persistenz der nukleären cccDNA (covalently closed circular DNA) des HBV. Die cccDNA ist folglich ein wichtiges therapeutisches Zielmolekül. Daher war es das Ziel dieser Arbeit, die Mechanismen der nicht-zytolytischen Reduktion der cccDNA durch Zytokine detaillierter aufzuklären.
Im ersten Teil der Arbeit zeigten Zytokin-Neutralisierungsversuche im Transwell, dass aktivierte T-Zellen die Zytokine Interferon-gamma (IFN-) und Tumornekrosefaktor-alpha (TNF-) sekretieren, welche den Verlust der cccDNA in HBV-infizierten Hepatomzellen ohne Zytolyse vermitteln. Diese Zytokine induzierten die Expression von APOBEC3A (A3A) und APOBEC3B (A3B), die nukleäre HBV-DNA desaminierten, und dabei essenziell für die Reduktion der cccDNA waren, wie Knockdown-Experimente belegten.
Im zweiten Teil wurde die Spezifität der cccDNA-Reduktion durch verschiedene Behandlungen untersucht: IFN-, TNF-, Interferon-alpha (IFN-) und Aktivierung des Lymphotoxin--Rezeptors (LTR). Die induzierte Desaminierung und Mengenreduktion betraf nur cccDNA, nicht aber episomale HBV-DNA (pEpi-H1.3). Da alle diese Behandlungen die Expression von A3A und A3B auslösen, wurden diese Desaminasen überexprimiert, was zu Hypermutationen auf der cccDNA führte. Außerdem zeigte Chromatin-Immunopräzipitation (ChIP), dass A3A und das HBV-Coreprotein mit der cccDNA assoziieren, was die Hypothese stützt, dass das HBV-Coreprotein die nukleären APOBEC3-Proteine zur cccDNA bringt.
Da die Expression von A3A zwar Desaminierung verursachte, jedoch allein nicht ausreichend für den Verlust der cccDNA war, strebte der dritte Teil der Arbeit die Identifizierung der involvierten Nuklease an. Eine Genexpressionsanalyse unter Behandlung mit IFN- ergab drei hochregulierte Kandidatennukleasen mit nukleärer Lokalisierung. ISG20 (interferon-stimulated gene product of 20 kDa), die am stärksten induzierte Nuklease, konnte in Lebergewebeproben von Patienten mit akuter, nicht aber chronischer, Hepatitis B angefärbt werden. ISG20-Knockdown verhinderte die Abnahme der cccDNA unter Interferonbehandlung. Überexpression von ISG20 zusammen mit A3A reichte aus, um die cccDNA ohne weitere Behandlung zu reduzieren.
Insgesamt führen die Ergebnisse dieser Arbeit zur Schlussfolgerung, dass die T-Zellzytokine IFN- und TNF- sowie IFN- und die Aktivierung des LTR zum nicht- zytolytischen Verlust der cccDNA, der Persistenzform des HBV, führen, wobei APOBEC3-Desaminasen und die Interferon-induzierte Nuklease ISG20 eine entscheidende Rolle spielen. Die gemeinsame Expression der Desaminase A3A und der Nuklease ISG20 reicht aus, um die cccDNA zu reduzieren. Diese Ergebnisse bilden eine solide Basis für die Weiterentwicklung der Therapien gegen chronische Hepatitis B.
1 Introduction
Despite an effective prophylactic vaccine, an estimated 257 million humans worldwide are suffering from chronic hepatitis B with the risk of developing liver cirrhosis or hepatocellular carcinoma, which cause more than 800,000 deaths per year (Naghavi et al. 2015, WHO 2017). A major problem in treatment of chronic hepatitis B is the viral persistence in infected liver cells. Therefore, this thesis deals with mechanisms of the control of viral persistence, induced by cytokines and without cytolysis.
1.1 Hepatitis B virus (HBV)
Since the present study focuses on molecular, cytokine-triggered mechanisms against HBV, the following chapters introduce the molecular biology of HBV, characteristics of HBV infection and therapy options.
1.1.1 Classification of HBV
Human HBV is the prototype member of the hepadnaviridae (Schaefer 2007), a family of small, enveloped DNA viruses. Members share tropism for liver tissue and a narrow host specificity (Modrow et al. 2010). Based on their ability to infect mammals or birds, hepadnaviridae are divided into the genera orthohepadnaviridae and avihepadnaviridae (Schaefer 2007). An example for orthohepadnaviridae is, beside human HBV, woodchuck hepatitis virus (Schaefer 2007, Summers et al. 1978), while duck HBV (DHBV) is representative for avihepadnaviridae (Mason et al. 1980, Schaefer 2007).
Since these DNA viruses replicate via an RNA intermediate, they are designated as para- retroviruses (Hu and Liu 2017).
HBV is subdivided phylogenetically into nine genotypes, A – I, based on a sequence divergence of more than 7.5 % across the whole genome between the groups. HBV genotypes have a diverse geographical distribution. For instance, genotype A appears in Africa, Europe and the Americas, genotypes B and C in Asia and genotypes F and H are prevalent in Southern and Central America. Genotype D is found worldwide, genotype G in parts of America and Europe, whereas genotype E is distributed in Western and Central Africa and genotype I in Asia (Kramvis 2014). A putative 10th genotype (“J”) was isolated from a single individual (Tatematsu et al. 2009). Further, HBV can be classified based on the epitopes of the surface protein into 9 different serological subtypes, which can be summarized in the four major serotypes ayw, ayr, adw and adr (Kramvis 2014).
1.1.2 Structure of HBV particles
Hepatitis B virions - also named “Dane particles” after their first visualisation - are enveloped particles of 42 nm in diameter (Dane et al. 1970). Figure 1 depicts their composition: The envelope consists of small (S), middle (M) and large (L) HBV surface proteins (HBs) in a lipid bilayer and engulfs a nucleocapsid (Nassal 2015). The icosahedral nucleocapsid is formed by 180 (T=3) or 240 (T=4) HBV core protein (HBc) subunits, which form dimers (Crowther et al. 1994). Inside, the partially double-stranded, relaxed-circular (rcDNA) genome of HBV is situated. The 5’ end of the minus strand of rcDNA is covalently bound to the viral polymerase protein (P protein) (Gerlich and Robinson 1980, Nassal 2015). Beside these infectious HBV particles, DNA-free subviral particles exist: 22 nm spheres and filaments, which consist of envelope proteins but lack nucleocapsid and rcDNA inside, as well as enveloped nucleocapsids without an rcDNA genome (Hu and Liu 2017).
1.1.3 HBV genome organisation
The HBV rcDNA genome is only 3.2 kb long and therefore tightly organized with overlapping open reading frames and each nucleotide having coding function. Figure 2 shows the arrangement of the open reading frames (Nassal 2015) encoding seven viral proteins: All three HBV surface proteins have the same carboxy-terminal domain (S), the additional parts of the M and L protein are N-terminal extensions named preS2 and preS1, respectively. HBV core protein (C) serves as subunit of the viral nucleocapsid as described above. The HBV core sequence with an additional N-terminal peptide (precore, preC) is proteolytically processed and secreted as hepatitis B e antigen (HBeAg) (Seeger and Mason 2015) and seems to have immunoregulatory functions (Chen et al. 2005). The P protein or polymerase protein functions as reverse transcriptase, RNaseH and primer during synthesis of the rcDNA genome (Seeger and Mason 2015). The non-structural HBV X protein (HBx; X) is needed for initiation and maintenance of viral transcription (Lucifora, Arzberger et al. 2011).
Figure 1: Structure of HBV. The envelope consists of small (S), middle (M) and large (L) surface proteins, embedded in a lipid layer. It encloses the nucleocapsid (core particle), which is composed of HBV core proteins, and the relaxed-circular DNA (rcDNA) genome of HBV, which is covalently linked to the viral polymerase (P) protein (Nassal 2015).
1.1.4 Replication cycle of HBV
The replication cycle of HBV is presented schematically in figure 3: To enter susceptible cells, viral LHBs attach to glycosaminoglycan side chains of cellular surface heparan sulphate proteoglycans (Schulze et al. 2007). Further, the myristoylated preS1 domain of LHBs interacts with sodium taurocholate cotransporting polypeptide (NTCP), which was identified as a functional receptor of HBV (Yan, Zhong et al. 2012). This multiple transmembrane transporter is expressed on primary hepatocytes and makes otherwise resistant hepatoma cell lines susceptible for HBV infection. For instance, human HepG2 cells support HBV infection after NTCP expression (Yan, Zhong et al. 2012). HBV entry into hepatocytes involves clathrin-dependent endocytosis (Huang et al. 2012). After uncoating and release of the genome-containing nucleocapsids into the host cell cytoplasm, nucleocapsids are translocated to the nuclear membrane. In the nuclear basket, the nucleocapsid shell disassembles and rcDNA is released into the nucleus (Schmitz, Schwarz et al. 2010). Nuclear rcDNA is completed to form the fully double- stranded, covalently closed circular DNA (cccDNA), which stays episomally in the nuclei of infected cells. cccDNA is the template for transcription by host RNA polymerase II of all viral RNAs, including subgenomic mRNAs for expression of HBs and HBx as well as the pregenomic RNA (pgRNA) and precore RNA. Precore RNA is translated, proteolytically processed and secreted as HBeAg whereas pgRNA is transmitted into P protein and HBV core protein. Additionally, pgRNA is reverse-transcribed by the viral P protein into rcDNA within the nucleocapsid (Nassal 2015, Seeger and Mason 2015).
Mature rcDNA-containing nucleocapsids are either transported back to the nucleus and increase cccDNA copy number or are enveloped and secreted, depending on the concentration of available envelope proteins at the endoplasmic reticulum (Modrow et al. 2010, Nassal 2015). Enveloped virions (Watanabe et al. 2007) and subviral filaments (Jiang et al. 2016) exit the cell via multivesicular bodies while subviral spheres are secreted via the endoplasmic reticulum and Golgi complex (Patient et al. 2007).
Figure 2: HBV genome organisation. Outer lines depict viral transcripts with transcription starts shown as white arrowheads, stands for the encapsidation signal on pregenomic RNA (pgRNA). Green arrows denote promotors, Enh I / Enh II transcriptional enhancers, DR1 / DR2 direct repeats, TP the terminal protein domain of P protein. HBV DNA is shown as rcDNA but transcriptional template is the covalently closed circular DNA (see replication cycle below).
Further details are given in the text (Nassal 2015).
Figure 3: Replication cycle of HBV. HBV attaches to heparan sulphate proteogylcans on the cell surface and interacts specifically with NTCP during entry. After uncoating, nucleocapsids are brought to the nucleus and rcDNA (relaxed-circular DNA) genomes are released into the nucleus.
There, rcDNA is completed to form cccDNA (covalently closed circular DNA), the viral persistence form. cccDNA serves as transcription template for all viral RNAs. pgRNA (pregenomic RNA) is encapsidated together with HBV polymerase (P protein) and reverse transcribed. Newly formed rcDNA-containing capsids are either transported back to the nucleus or enveloped at the endoplasmic reticulum and released via multivesicular bodies (Ko et al. 2017).
The process of reverse transcription is depicted in figure 4: P protein binds to the stem- loop structure of pgRNA and mediates co-packaging of pgRNA and P protein into newly forming nucleocapsids, whereby the carboxy-terminal domain of HBV core protein is needed. A tyrosine residue in the terminal protein domain of P protein provides the 3’ OH
group for priming reverse transcription of minus-strand DNA, resulting in covalent binding (Nassal 2015, Zlotnick et al. 2015). After synthesis of some nucleotides of the minus- strand DNA, P protein and covalently bound DNA are translocated to the 3' end of pgRNA to hybridize with the complementary sequence of direct repeat 1 (DR1).
Extension yields whole minus-strand DNA, while P protein degrades pgRNA through its RNaseH activity except for a 5' residue. When proper rcDNA is formed, this RNA oligomer is translocated to the complementary direct repeat 2 (DR2) at the 5' end of the minus-strand DNA, where it primes plus-strand DNA synthesis; the RNA primer stays bound to the 5’ end of the plus strand in the end. However, if primer translocation and circularization fail, double-stranded linear DNA may be formed. A final template switch circularizes minus-strand DNA within the nucleocapsid and provokes that plus-strand DNA synthesis spans the gap caused by the covalently bound P protein at the minus strand. The plus strand remains incomplete forming the partially double-stranded rcDNA (Modrow et al. 2010, Nassal 2015, Zlotnick et al. 2015).
Figure 4: Reverse transcription. (A) Transcription of pgRNA from cccDNA. (B) Binding of P protein to pgRNA, start of transcription of minus-strand DNA and minus-strand template switch.
(C) Minus-strand DNA elongation, RNase H digestion of pgRNA. (D) RNA primer translocation.
(E) Start of plus-strand DNA synthesis. (F) Circularization by template switch. (G) Plus-strand DNA elongation. More details are given in the main text (Zlotnick et al. 2015).
1.1.5 Formation and persistence of HBV cccDNA
The cccDNA is the persistence form of HBV and, thus, a major therapeutic target in chronic hepatitis B. It is formed from rcDNA in the nucleus of infected cells. The rcDNA differs from cccDNA by having a covalently linked P protein, an RNA primer at the 5’ end of the plus-strand DNA, terminal redundancies at the minus-strand DNA, an incomplete plus strand and it is nicked. Because of the distinct molecular properties of rcDNA and cccDNA, several enzymatic steps are supposedly involved in the conversion but only few details of this process are known so far (Nassal 2015). Königer et al. provided evidence for the involvement of tyrosyl-DNA-phosphodiesterase (TDP) 2 in generation of cccDNA.
Viral P protein is linked to rcDNA by a tyrosyl-DNA phosphodiester bond and resembles thereby cellular topoisomerase-DNA adducts, which are repaired by cellular TDP1 or TDP2 enzymes. Human TDP2 can release P protein from HBV rcDNA in vitro (Königer et al. 2014). Furthermore, Qi et al. demonstrated that inhibition of host DNA polymerase (POLK) impairs cccDNA generation, an effect which can be rescued partially by ectopic expression of POLK. Results indicated that POLK is involved in cccDNA formation during HBV infection by repairing the gap of rcDNA (Qi et al. 2016).
However, further steps in cccDNA generation remain elusive.
After formation of cccDNA, it persists as episome in the nucleus of the infected cell and gets organized into a viral minichromosome, i.e. it acquires a chromatin-like structure by nucleosomal packaging (Levrero et al. 2009, Nassal 2015), as shown in figure 5. The HBV minichromosome consists of histones H3 and H2B and to lower levels of histones H4, H2A and H1 (Bock et al. 2001). Additionally, HBV core protein binds to cccDNA (Bock et al. 2001, Guo, Li et al. 2011, Pollicino et al. 2006), which results in a reduction of the nucleosomal spacing (Bock et al. 2001). Histone posttranslational modifications favouring active transcription are enriched on cccDNA and repressive histone marks are underrepresented (Tropberger et al. 2015), but histone modifications are changeable.
Modification of histone methylation patterns influences transcription from cccDNA (Rivière et al. 2015) as well as acetylation of cccDNA-bound H3 and H4 histones modulates HBV replication (Pollicino et al. 2006). Also, the viral HBx was shown to enable transcription from cccDNA by preventing deacetylation of cccDNA-attached histones (Belloni et al. 2009, Lucifora, Arzberger et al. 2011) and to avoid transcriptional repression of cccDNA by modification of histone methylation (Rivière et al. 2015).
Moreover, interferon-alpha (IFN-) treatment leads to hypoacetylation of cccDNA-bound histones and, thus, inhibition of transcription from cccDNA (Belloni et al. 2012, Tropberger et al. 2015). Furthermore, cccDNA itself can be methylated and hypermethylation correlates with decreased transcription and HBV replication (Guo, Li et al. 2011, Guo et al. 2009). The epigenetic features of cccDNA, which determine its permissiveness for transcription, are summarized in figure 6.
Few cccDNA copies per nucleus of infected cell are reported. In livers of chronically infected ducks, a mean of 10 cccDNA copies/cells were detected, whereby copy numbers distributed between one and 17 cccDNA copies/cell (Zhang et al. 2003). In human, HBV-infected HepG2-NTCP cells, cccDNA copy number was estimated to 2.4 copies/cell (Tropberger et al. 2015). Half-life of cccDNA in DHBV-infected ducks was determined at 35 to 57 days (Addison et al. 2002), in HBV-infected human HepG2-NTCP cells at approx. 30 days (Ko et al. 2017). Cell division of in vivo proliferating HBV-infected hepatocytes can lead to cccDNA loss (Lutgehetmann, Volz et al. 2010), but in cell culture systems cccDNA endures several rounds of cell division (Ko et al. 2017). Whether reimport of rcDNA-containing nucleocapsids from the cytoplasm refills the cccDNA pool, depends on the model system. In DHBV infection, cccDNA can recycle efficiently (Köck et al. 2010) but dHepaRG cells, differentiated human hepatoma cells, favour long-term persistence of HBV without cccDNA amplification (Hantz et al. 2009). Due to the stability of the cccDNA pool in dHepaRG cells, this cell culture system was preferentially used in this thesis.
Figure 5: Electron microscopy of HBV cccDNA forms. (1) In vitro reconstituted HBV nucleoprotein complexes, (2) HBV nucleoprotein complexes under physiological ionic conditions and (3) in low salt buffer showing “beads-on-a- string” appearance. Scale bar: 0.2 µm (Bock et al. 2001).
Figure 6: Epigenetic features of cccDNA. cccDNA is associated with nucleosomes consisting of histones H2A, H2B, H3, H4 and H1. Histone post-translational modifications (PTM) and DNA methylation (me) regulate the epigenetic permissiveness of cccDNA.
The HBx modulates the epigenetic status of cccDNA and, thus, transcription by RNA polymerase II (RNA pol II) (Nassal 2015).
1.1.6 HBV infection and therapy
Transmission of HBV occurs via blood or blood products, sexual contacts and from mother to child during birth (Modrow et al. 2010). Infection with HBV is often asymptomatic in adults, only one third shows symptoms of an acute, icteric hepatitis and 0.5 – 1 % of infected adults develop a fulminant hepatitis with liver failure. 5 – 10 % of HBV-infected adults develop chronic hepatitis (RKI 2015). In contrast, children up to six months of age develop chronic hepatitis B in 90 % of infection cases. Besides young age, the risk of developing chronic hepatitis B increases to 30 – 90 % under immune suppression (RKI 2015, Schweitzer et al. 2015).
HBV is a non-cytopathic virus, i.e. liver damage and viral control are mediated by the immune system. Hepatitis B surface antigen (HBsAg), that mainly consists of spherical subviral particles, appears two to ten weeks after HBV infection. In acute hepatitis B, alanine aminotransferase (ALT) concentrations as indicator of liver inflammation raise one to two weeks after the appearance of HBsAg, simultaneously to appearance of symptoms and anti-HBV-core antibodies. Anti-HBsAg antibodies raise several weeks later, approximately at the time point when HBsAg is cleared from serum in people who recover. In this case, ALT levels normalize and viral marker and symptoms disappear. If HBsAg persists for more than six months, HBV infection is considered as chronic.
Following a high replicative phase without obvious liver disease, HBV DNA levels decrease and ALT levels increase in the “immune clearance” phase of chronic HBV infection. The immune response, however, fails to clear the virus. When HBeAg seroconversion takes place, HBV DNA levels are lowered and when ALT concentrations normalize, transition into the inactive phase takes place. In 20 – 30 % of patients, reactivation will occur with increase of HBV DNA and/or ALT levels after HBeAg seroconversion. These patients have an increased risk to develop liver cirrhosis and hepatocellular carcinoma (Trépo et al. 2014).
Currently, seven antivirals are approved for therapy of chronic hepatitis B: conventional or PEGylated IFN- and the nucleoside or nucleotide analogues entecavir, lamivudine, tenofovir, adefovir and telbivudine. IFN- has antiviral as well as immunomodulatory effects. A big disadvantage of IFN-, however, are the side-effects, such as influenza- like symptoms, fatigue, bone marrow suppression, depression and unmasking of autoimmune diseases (Trépo et al. 2014). Treatment with PEGylated IFN- is rather inefficient with response rates below 10 % defined by HBsAg seroconversion (Janssen et al. 2005).
Nucleos(t)ide analogues inhibit viral reverse transcription of pgRNA into rcDNA and affect therefore HBV replication late in the viral replication cycle. They are not directly targeting the persistence form cccDNA, which makes viral relapse common after treatment stop (Revill et al. 2016, Trépo et al. 2014). Nucleos(t)ide analogue treatment suppresses HBV but cannot eradicate the virus completely. Therefore, further research is needed to cure chronic HBV infection. The current opinion in the field is, that a
“functional cure” might suffice, which is characterized by HBsAg loss and HBsAg seroconversion, undetectable serum HBV DNA amounts and transcriptionally inactive cccDNA, enabling to stop treatment (Revill et al. 2016).
1.2 Antiviral T-cell immunity
A proper adaptive immune response early in HBV infection is essential for an efficient control of the virus infection. Thus, the following chapters focus on the antiviral role of T cells in HBV infection, their cytotoxic and non-cytolytic functions.
1.2.1 T-cell responses in acute and chronic hepatitis B
The initial phase after HBV infection, when HBV DNA is not yet or only weakly detected, is followed by an exponential increase of HBV replication. In acute HBV infection, HBV- specific CD4 and CD8 T-cell responses are detected (Bertoletti and Ferrari 2012). CD4 helper and CD8 cytotoxic T cells react to epitopes of HBV core protein, P protein and envelope proteins (Said and Abdelwahab 2015). T-cell responses in acute and self- limiting HBV infection are typically multispecific, strong and polyfunctional (Bertoletti and Ferrari 2012, Said and Abdelwahab 2015). Additionally, T-cells secrete cytokines and contribute to the antiviral effect against HBV without liver cell destruction (Guidotti et al.
1999). When acute HBV infection is controlled, T cells mature into memory cells, marked by increased expression of CD127 and decreased expression of programmed death (PD)-1 on HBV-specific CD8 T cells (Boettler, Panther et al. 2006).
In chronic hepatitis B, HBV persistence is linked to impaired functions of HBV-specific CD4 and CD8 T cells. High viremia correlates with suppressed HBV-specific T-cell responses and T cells in the liver are less functional than in the periphery (Bertoletti and Ferrari 2012). High viral antigen loads and tolerogenic features of hepatocytes might contribute to a weak, oligoclonal and exhausted T-cell response in chronic hepatitis B (Bertoletti and Ferrari 2012, Protzer et al. 2012). Exhaustion (i.e. functional inactivation) is driven by increased negative co-regulation, mediated by e.g. PD-1 expression, which can inhibit antiviral immunity in the liver (Iwai et al. 2003). Further, virus-specific cytotoxic T cells are depleted during chronic HBV infection, in part by enhanced susceptibility to apoptosis (Lopes et al. 2008).
1.2.2 Cytotoxic T-cell response
As part of the adaptive immune system, cytotoxic T cells (also: cytotoxic T lymphocytes, cytolytic T cells, CD8 T cells) can recognize and eliminate cells presenting foreign antigen, e.g. virus-infected cells. Cytotoxic T cells are primed in lymphoid organs by professional antigen presenting cells (APCs) or dendritic cells. Activation of naïve T cells
by APCs requires contact with an antigen via their specific T-cell receptor (TCR), CD8 or CD4 co-receptor binding, co-stimulation by interaction of T-cell CD28 with its ligand B7 on the APC and cytokine stimulation for T-cell proliferation and differentiation. T cells with CD8 receptors interact with MHC I (major histocompatibility complex I) molecules.
When a cell is infected with a virus, its MHC I molecule can get loaded with viral antigen processed into a peptide of approximately nine amino acids allowing recognition of the peptide/MHC I complex by the TCR. After clonal expansion, cytotoxic T cells migrate from lymph nodes to the infected tissue, where they recognize foreign antigen and elicit their effector functions without the need for co-stimulation (Murphy and Weaver 2016).
CD8 T cells have two major pathways to perform their cytotoxic effector functions: They can release granzyme B and perforin. Perforin enables entry of granzyme B into the cytoplasm of the target cells and granzyme B induces apoptosis. Additionally, the membrane-bound Fas ligand (FasL) binds to the death receptor Fas (CD95), which triggers activation of a caspase cascade resulting in apoptosis (Thome and Tschopp 2001).
1.2.3 Non-cytolytic T-cell functions
Beside target cell killing, CD8 T cells secrete cytokines and elicit non-cytolytic antiviral functions. Evidence for it comes from studies with HBV transgenic mice. HBV-specific cytotoxic T cells suppress HBV gene expression through the cytokines interferon-gamma (IFN- and tumour necrosis factor-alpha (TNF-) in a non-cytolytic manner, which is stronger than the cytolytic effect (Guidotti et al. 1994). Also, HBV-specific cytotoxic T cells from perforin-deficient mice were still able to inhibit HBV replication without inducing liver disease (Guidotti et al. 1996). In acute HBV infection in chimpanzees, HBV DNA in the liver was diminished before liver disease and CD8 T cell infiltration reached their maxima, indicating that non-cytolytic mechanisms contribute to the purging of HBV cccDNA from infected cells (Guidotti et al. 1999). Similarly, Wieland, Spangenberg et al.
showed in HBV-infected chimpanzees that cccDNA is reduced initially in a non-cytolytic manner. In this phase, cccDNA declined in parallel to appearance of IFN- producing CD8 T cells in the liver, while HBV-core-positive hepatocytes persisted longer (figure 7) (Wieland, Spangenberg et al. 2004). In vitro experiments further evidenced that HBV- specific CD8 T cells can control HBV replication without cytolysis via IFN- and TNF-
and that these non-cytolytic mechanisms contribute importantly to the antiviral function of CD8 T cells (Phillips et al. 2010).
1.3 Antiviral effects of cytokines
As inflammatory mediators, cytokines are involved in antiviral defence. There are more than 60 different cytokines (Murphy and Weaver 2016), of which the four important ones for this thesis are described in the following chapters in more detail.
1.3.1 Interferon-alpha (IFN-)
Interferons are small, secreted proteins and have been discovered as substances that interfere with influenza virus infection. The largest class of interferons are type I interferons, which compromise IFN- and interferon-beta (IFN-) among others. Nearly all cells can produce IFN- but during infection it is mainly produced by plasmacytoid dendritic cells. Type I interferons bind to a heterodimeric receptor consisting of an IFN-
receptor 1 (IFNAR1) and IFNAR2 subunit. Intracellular signalling is mediated by phosphorylation of Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) leading to phosphorylation and heterodimerisation of signal transducers and activators of transcription 1 (STAT1) and STAT2 proteins. After association with interferon-regulatory factor 9 (IRF9), the whole complex translocates to the nucleus to activate transcription of interferon-stimulated genes (ISGs) (figure 8) (Schneider et al. 2014). ISGs with antiviral activity against HBV are, for instance, myxoma resistance protein 1 (MxA), which inhibits HBV replication at posttranscriptional level (Gordien et al. 2001), apolipoprotein B editing complex 3 (APOBEC3) deaminases (Janahi and McGarvey 2013) and interferon-stimulated gene product of 20 kDa (ISG20) (Leong, Funami et al.
2016, Liu et al. 2017, Ma et al. 2016).The latter two are described in the chapters below in more detail.
Type I interferons can modulate innate and adaptive immune responses, e.g. by stimulating effector functions of natural killer cells, cytotoxic T cells and macrophages, by enhancing antigen presentation and induction of antibody production. Further, they can inhibit cell division and stimulate proliferation of memory T cells (Guidotti and Chisari 2001). Beside their immunomodulatory functions, type I interferons elicit direct antiviral activity against a broad range of viruses and IFN- was shown to inhibit HBV on several Figure 7: Decline of cccDNA in acutely HBV- infected chimpanzee. While cccDNA decreases after week 8, HBV core protein (HBcAg) starts to diminish after week 9, indicating that the loss of cccDNA is not reflecting target cell lysis but non- cytolytic effects (Wieland, Spangenberg et al.
2004).
distinct levels. For instance, cells treated with IFN- release factors inhibiting HBV entry by competing with viral binding to heparan sulphate proteoglycans (Xia et al. 2017a).
IFN- can also lead to epigenetic silencing of HBV cccDNA, thereby inhibiting viral transcription (Belloni et al. 2012). Further studies confirmed the IFN--triggered HBV repression by modulation of histones associated with cccDNA (Tropberger et al. 2015).
Additionally, IFN- leads to decay of HBV nucleic-acid containing capsids (Xu et al.
2010).
1.3.2 Interferon-gamma (IFN-)
IFN- is the only representative of type II interferons. It is produced by cells of the immune system but nearly all cell types express the IFN- receptor (IFNGR) and can, thus, respond to it. Binding of IFN- to two IFNGR1 subunits causes additional binding of two IFNGR2 subunits. Following receptor activation, JAK1 and JAK2 kinases are phosphorylated resulting in dimerization of two phosphorylated STAT1 proteins. These homodimers enter the nucleus to induce target ISG transcription (figure 9) (Schneider et al. 2014). As IFN-does, IFN- induces the expression of APOBEC3 proteins (Janahi and McGarvey 2013) and ISG20 (Gongora et al. 1997). More details on their antiviral role against HBV are given in the chapters below.
Figure 8: Type I interferon (IFN) signalling.
Type I interferons bind to their receptor on the cell surface inducing JAK/STAT signalling.
Formation of the complex interferon-stimulated gene factor 3 (ISGF3) enables target gene expression after translocation to the nucleus and binding to interferon-stimulated response elements (ISRE). Similar signalling is induced by type III interferons binding to interleukin-10 receptor 2 (IL-10R2) and interferon-lambda receptor 1 (IFNLR1) (Schneider et al. 2014).
Antiviral T-cell cytokines, such as IFN-, can control viral infection indirectly, by immunoregulatory activity, enhancing antigen presentation, triggering homing of T cells to the infected tissue or stimulating the cytolytic function of effector cells of the innate or adaptive immune response (Guidotti and Chisari 2001). IFN- is also capable of controlling HBV replication directly and can induce non-cytolytic HBV DNA decline (Guidotti et al. 1999, Phillips et al. 2010, Wieland, Spangenberg et al. 2004). Moreover, IFN- was shown to destabilize HBV RNAs (Heise et al. 1999), to inhibit viral protein translation by tryptophan deprivation (Mao et al. 2011) and to accelerate the decay of nucleic-acid containing HBV capsids (Xu et al. 2010).
1.3.3 Tumour necrosis factor-alpha (TNF-)
TNF was originally found as tumour-destroying cytokine, where it got its name from. The TNF superfamily includes many members, e.g. TNF-, FasL, CD40 ligand, OX40 ligand and lymphotoxins. TNF- is secreted by activated T cells, monocytes, natural killer cells, mast cells, B cells and Kupffer cells in the liver. IFN-can increase its expression.
Secreted TNF- binds preferentially to TNF receptor 1 (TNFR1), which is expressed on all cell types despite erythrocytes. The second receptor TNFR2 is inducible and expressed particularly on endothelial and hematopoietic cells (Valaydon et al. 2016). In general and depending on cell type, status of the cell and cell cycle, TNFR1 activation leads to induction of apoptosis. This involves the adaptor proteins TNFR1-associated death domain (TRADD) and Fas-associated death domain (FADD) and is triggered by a Figure 9: Type II interferon signalling. Type II interferons bind to their receptors triggering the JAK/STAT pathway. Phosphorylated STAT1 homodimers build the IFN- activation factor (GAF), that translocates to the nucleus, where it binds to gamma-activated sequence (GAS) promoter elements to induce target gene expression (Schneider et al. 2014).
caspase cascade. In contrast, TNFR2 activation can promote cell survival. TNFR2 signalling involves cellular inhibitor of apoptosis protein (cIAP) and leads to nuclear translocation of the transcription factor NF-B resulting in transcription of pro-survival genes (figure 10) (Faustman and Davis 2010, Valaydon et al. 2016).
To fulfil its antiviral function against HBV, TNF- can induce the non-cytolytic disruption of HBV nucleocapsids via NF-B signalling (Biermer et al. 2003). Furthermore, TNF-
destabilizes HBV RNAs together with IFN- (Guidotti et al. 1996). As T-cell cytokine, it mediates the non-cytolytic control of HBV replication (Phillips et al. 2010).
Polymorphisms in the TNF- gene are associated with the outcome of HBV infection, such as the 238A allele was linked with an increased risk to develop chronic hepatitis B in European populations (Zheng et al. 2012). Moreover, anti-TNF- therapy, which is applied for treatment of inflammatory arthritis, can result in HBV reactivation in patients with chronic HBV infection who do not receive antiviral prophylaxis (Ye, Zhang et al.
2014).
1.3.4 Lymphotoxins (LT)
Lymphotoxins are cytokines belonging to the TNF superfamily that are involved in lymph- node development (Murphy and Weaver 2016). They are expressed by T cells, B cells, natural killer cells and lymphoid tissue-inducer cells. Lymphotoxin-beta (LT) is a transmembrane protein and forms membrane-anchored heterotrimers together with lymphotoxin-alpha (LT), i.e. LT12and LT21. Matrix metalloproteases can cleave these heterotrimers from the cellular surface, however. In contrast, LT is secreted directly as soluble homotrimer. LT and LT21 activate TNFR1 or TNFR2, which are described above. In contrast, LT12 signals via LTR. LTR stimulation triggers signalling via canonical or non-canonical NF-B pathway. In the canonical NF-B Figure 10: TNF- induced pathways in hepatocytes. Recruitment of cIAP (cellular inhibitor of apoptosis) after receptor binding leads to upregulation of cell survival proteins. If cIAP is absent, cell death is induced (Valaydon et al. 2016).
signalling pathway, the complex of NEMO/IKK/IKK is activated leading to phosphorylation and proteasomal degradation of the inhibitor of B (IB). This allows the heterodimer p50/RelA to enter the nucleus and induce gene expression involved for example in inflammation and cell proliferation. In contrast, the non-canonical signalling activates NF-B-inducing kinase (NIK), which leads to phosphorylation of a homodimeric IKK complex. This results in phosphorylation and proteasomal degradation of p100 into p52, which enters the nucleus as a heterodimer with RelB triggering target gene expression, important for instance in lymph-node development or B-cell survival (figure 11) (Wolf et al. 2010).
Lymphotoxins and LTR were reported to be upregulated in HBV-induced hepatitis and hepatocellular carcinoma and sustained lymphotoxin signalling seems to be involved in hepatitis-induced carcinoma (Haybaeck, Zeller et al. 2009). However, depending on the cell type, LTR agonisation can inhibit tumour growth in human colon carcinoma, mammary carcinoma and soft tissue sarcoma cells (Hu, Zimmerman et al. 2013).
Figure 11: Canonical and non-canonical NF-B signalling induced by lymphotoxins. Many agonists induce the canonical NF-B pathway, here TNF and LT3 are shown as examples (left part). LT12 binding triggers non-canonical NF-B signalling (right part) but can also induce the canonical NF-B pathway (Wolf et al. 2010).
1.3.5 APOBEC3 deaminases as cytokine-induced effector proteins
APOBEC3 enzymes elicit antiviral activity against a broad range of DNA viruses and retroviruses (Stavrou and Ross 2015). These proteins are a family of cytidine deaminases converting cytosines to uracils in viral DNA (Janahi and McGarvey 2013).
Several members of the APOBEC3 family can mutate HBV in this way. For instance, APOBEC3G (A3G) was shown to induce hypermutations on HBV rcDNA (Kitamura et al. 2013). In contrast to the cytoplasmic A3G, APOBEC3B (A3B) can locate in both cytoplasm and nucleus and it was reported to hypermutate HBV genomes too (Bonvin et al. 2006). A3B and APOBEC3A (A3A) can deaminate single-stranded DNA during replication (Hoopes, Cortez et al. 2016). A3A was reported to be involved in the degradation mechanism of foreign DNA, as depicted in figure 12: Interferon induces expression of A3A, which deaminates foreign DNA generating a substrate for uracil DNA glycosylase (UNG) 2. After uracil excision, apurinic/apyrimidinic site endonuclease (APEX) might possibly function as nuclease causing DNA degradation (Stenglein et al.
2010).
Figure 12: Degradation of foreign DNA involving deamination by A3A.
Interferon-induced A3A deaminates foreign DNA leading to uracil excision by UNG2 (uracil DNA glycosylase 2) and DNA digestion possibly by APEX (apurinic/ apyrimidinic site endonuclease) (Stenglein et al. 2010).
1.3.6 ISG20 as interferon-induced effector nuclease
The interferon-induced ISG20 (Gongora et al. 1997) or HEM45 (HeLa Estrogen Modulated, band 45) (Pentecost 1998) is a 3’ to 5’ exonuclease, that can degrade single- stranded RNA and DNA (Nguyen et al. 2001). It belongs to the DEDDh subgroup of the DEDD exonuclease superfamily, which is characterized by three conserved aspartate (D), one conserved glutamate (E) and one conserved histidine (H) residue, and has three distinct exonuclease motifs named Exo I, Exo II and Exo III (Degols et al. 2007, Moser et al. 1997). ISG20 localizes to the nuclei and the cytoplasm of hepatocytes during response to IFN- treatment (Lu et al. 2013).
ISG20 exerts antiviral activity against several viruses (Zheng et al. 2017), e.g. vesicular stomatitis virus, influenza virus, encephalomyocarditis virus (Espert et al. 2003) and human immunodeficiency virus type 1 (HIV-1) (Espert, Degols et al. 2005). Replication of HIV-1 was delayed by ISG20 when a catalytically active form was expressed (Espert, Degols et al. 2005). Recent studies showed that ISG20 expression itself can block HBV replication by degrading HBV RNA via its exonuclease activity (Leong, Funami et al.
2016, Liu et al. 2017, Ma et al. 2016). Through its binding to the stem-loop structure of HBV RNA, ISG20 can prevent pgRNA encapsidation even in its catalytically inactive form (Liu et al. 2017), thus providing several antiviral modes of action.
1.4 Aims of the study
So far, no curative treatment is available for chronic hepatitis B. A major problem in chronic HBV infection is the viral persistence mediated by the HBV cccDNA form in the nucleus of infected hepatocytes. Therefore, cccDNA is an important therapeutic target.
Beside approaches which lead to direct killing of HBV-infected cells, non-cytolytic antiviral strategies targeting cccDNA gain increasing interest. The overall aim of this thesis was, thus, to obtain a detailed picture of the molecular mechanisms of non- cytolytic cccDNA loss through antiviral cytokine activity.
The first question addressed in this thesis was whether T-cell cytokines can reduce cccDNA in human hepatocytes without cytolysis, and by which mechanism this is achieved. A transwell co-culture system should be established to avoid killing through direct contact of T cells and HBV-infected target cells and to investigate the cytokine secretion of activated T cells together with the cytokine effects on HBV DNA and protein expression.
The aim of the second part of the thesis was to figure out whether the cytokine-induced DNA decline is specific for cccDNA or whether it can target any episomal DNA. For that purpose, a hepatoma cell line replicating HBV from an episomal construct should be studied regarding cytokine-induced deamination and decay of cccDNA and episomal DNA as well as protein interactions with cccDNA should be examined.
The third part of the thesis aimed at identifying the nuclease that is triggering cccDNA decay under treatment with cytokines IFN- and IFN-. To this end, gene expression under interferon treatment in human hepatoma cells should be investigated and functional studies should be carried out to ascertain the nuclease involved in interferon- induced cccDNA reduction.
2 Results
According to the aims of the thesis, this part deals with three aspects of the control of HBV persistence, namely the non-cytolytic reduction of cccDNA by T-cell cytokines, the specificity of the treatment-induced cccDNA decline and the nuclease involved in interferon-triggered cccDNA loss in this mechanism.
2.1 Non-cytolytic reduction of HBV cccDNA by T-cell cytokines IFN- and TNF-
In animal models, it has been suggested that T-cell cytokines contribute non-cytolytically to HBV clearance in acute, self-limiting hepatitis B (Guidotti et al. 1999, Wieland, Spangenberg et al. 2004). Further studies in vitro confirmed that T-cell cytokines IFN-
and TNF- are involved in the inhibition of HBV without cytolysis (Phillips et al. 2010).
This part of the thesis, thus, examined the non-cytolytic effect of these T-cell cytokines on HBV cccDNA in human hepatoma cells focusing thereby on the mechanism of the reduction of cccDNA, the HBV persistence form.
2.1.1 Activated T cells secrete IFN- and TNF- and induce cccDNA loss
First, to investigate non-cytolytic effects of T-cell cytokines directed against HBV in human hepatoma cells, a transwell co-culture system was established. This allows a separation of non-cytolytic T-cell functions from direct killing by cytotoxic T cells:
Hepatoma cells expressing HBsAg on their surface (Huh7-S cells) or control cells (Huh7 cells) were seeded into the upper transwell chamber. On top of these cells, T cells were added which have a chimeric antigen receptor recognizing HBsAg (S-CAR) (Bohne et al. 2008). S-CAR T cells are activated by HBsAg on Huh7-S cells (figure 13A, left). Here, these S-CAR T cells were used as a model for HBV-specific T cells, that can be stimulated by the viral HBs leading to cytokine secretion and control of HBV replication (Bohne et al. 2008, Krebs, Böttinger et al. 2013). Activated T cells secreting cytokines were then transferred within the transwell-chamber to a cell culture vessel, where HepaRG cells were grown, differentiated and infected with HBV before. The secreted T-cell cytokines could pass through the pores of the transwell membrane and reached the HBV-infected target cells without direct contact of T cells and target cells. Thereby, cytokine-mediated, non-cytolytic effects of T cells on HBV could be studied (figure 13A, right) (Xia, Stadler et al. 2016).
In accordance with chimpanzee studies (Guidotti et al. 1999, Wieland, Spangenberg et al. 2004), we could confirm that patients with acute hepatitis B have elevated serum IFN- and TNF- compared to patients with chronic hepatitis B or healthy donors (Xia, Stadler et al. 2016). Therefore, the secretion kinetics of IFN- and TNF- by activated T
cells were determined first in the transwell system. ELISA experiments showed that only S-CAR T cells which were activated by co-culture with Huh7-S cells led to secretion of detectable amounts of IFN- and TNF- To determine the T-cell activation kinetics, supernatants from independent samples were analysed after two, four, eight, 16 and 24 hours of antigen stimulation. The results obtained by ELISA showed, that T cells started secretion of detectable amounts of IFN- and TNF- after a minimum of 16 hours of antigen contact (figure 13B).
Transwells with activated S-CAR T cells for 16 hours were then transferred to HBV- infected dHepaRG cells. To test the minimal incubation time of activated T cells with target cells, which is necessary to get a loss of HBV cccDNA, transwells were removed after six, 12, 24, 36 or 48 hours of incubation. At the same time, cell culture supernatants were exchanged to remove secreted cytokines, so that stimulation of the HBV-infected target cells by cytokines was limited to the indicated time frames between six and 48 hours. HBV-infected dHepaRG cells were rested subsequently under standard culture conditions for a total of seven days to allow cytokine-induced effectors to elicit their antiviral functions. Results revealed that a 12-hour incubation time with activated T cells was sufficient to reduce cccDNA levels, reaching the maximal effect after 24 hours (figure 13C). Slightly delayed, also HBeAg (figure 13D) and total intracellular HBV DNA (figure 13E) were reduced by activated S-CAR T cells after 24 to 48 hours of incubation time.
In summary, this shows that activated T cells secrete IFN- and TNF- and induce efficiently the reduction of HBV cccDNA, total intracellular HBV DNA and HBeAg levels (Xia, Stadler et al. 2016).
Figure 13: Cytokine secretion and non-cytolytic antiviral function of activated T cells. (A) HBsAg-specific (S-CAR) T cells were added in a transwell with either Huh7 or Huh7-S cells.
Huh7-S cells activated S-CAR T cells by expression of HBsAg on the cellular surface, leading to
cytokine secretion. Transwells were transferred to cell culture vessels, where HBV-infected differentiated HepaRG (dHepaRG) cells were cultivated. T-cell cytokines could pass through the porous membrane of the transwell and elicit non-cytolytic, antiviral effects, whereby the transwell avoided direct contact of T cells with infected dHepaRG cells. (B) S-CAR T cells were activated by Huh7-S cells or kept with Huh7 cells as control for indicated time frames. Supernatants taken from different wells at each time point were analysed by ELISA to determine kinetics of IFN- and TNF- secretion (n=3). (C) After 16 hours of activation, T cells within the transwells were transferred to HBV-infected dHepaRG cells (time point 0) and left there for the indicated incubation time to elicit their non-cytolytic functions. Amounts of cccDNA in HBV-infected dHepaRG cells, (D) HBeAg and (E) intracellular HBV DNA were determined at day seven by qPCR relative to PRNP (prion protein gene) or ELISA, respectively (n=3). Statistical analysis:
Student’s unpaired t-test with Welch’s correction (ns: not significant, p > 0.05); nd: not detectable (Xia, Stadler et al. 2016).
2.1.2 Decline of cccDNA is mediated by T-cell cytokines IFN- and TNF-
To ascertain the cytokines that mediate the non-cytolytic antiviral activity of T cells, neutralizing antibodies were applied in the transwell co-culture system. In this experimental setup, S-CAR T cells were stimulated by purified HBs (also termed HBsAg), which was used for coating of transwells, or kept without HBsAg as control. Stimulation of T cells without cytokine neutralization (no neutralizing antibodies) reduced cccDNA levels to approximately 20 % of the control level (without T-cell activation). IFN-
neutralization rescued about two-thirds of this cccDNA decline. TNF- neutralizing antibodies had a minor but still significant effect on blocking the reduction of cccDNA triggered by activated T cells. Both IFN- and TNF- neutralizing antibodies together restored cccDNA levels to more than 80 % of control (figure 14A). Analogously, IFN- or TNF- neutralization impaired HBeAg reduction and combination of both neutralizing antibodies restored HBeAg levels efficiently to over 80 % of control level (figure 14B) (Xia, Stadler et al. 2016).
As further potential T-cell effector cytokines (Murphy and Weaver 2016), lymphotoxins were neutralized by antibodies. Baminercept is a recombinant LTR protein that can block LT12 and LT21 signalling. Etanercept as a recombinant TNF-receptor p75 protein can neutralize TNF- and LT3 signalling but can also block receptor binding of LT12 and LT21 cytokines. Baminercept addition to stimulated T cells in the transwell system did not alter cccDNA nor HBeAg levels. Etanercept blocked T-cell- mediated reduction of cccDNA and HBeAg to a small but significant extent. However, IFN- neutralization was still responsible for the major effects on restoration of cccDNA and HBeAg levels (figure 14C-D).
Altogether, neutralization experiments confirmed that IFN- and TNF-are the key factors of cytokine-triggered, non-cytolytic cccDNA decline through activated T cells (Xia, Stadler et al. 2016).
Figure 14: Neutralization of T-cell derived cytokines. (A) HBsAg-stimulated S-CAR T cells were kept in the transwell system and antibodies for neutralizing IFN- (IFN- NeuAb) and TNF-
(TNF- NeuAb) were added to the cell culture medium. cccDNA levels from HBV-infected dHepaRG cells and (B) HBeAg levels were determined (n=3) (cooperation with Yuchen Xia).
(C, D) Analogously, antibodies neutralizing LTR-binding cytokines (Baminercept) or lymphotoxins and TNF- (Etanercept) were applied in the transwell setting and (C) cccDNA and (D) HBeAg from HBV-infected dHepaRG cells were measured after seven days of transwell co- culture (n=3). No NeuAb: HBsAg-stimluated S-CAR T cells without application of neutralizing antibodies. No HBsAg: Control samples with unstimulated S-CAR T cells. Statistical analysis:
Student’s unpaired t-test with Welch’s correction (ns: not significant, p > 0.05) (Xia, Stadler et al.
2016).
